1. Field
The present disclosure relates to methods of detecting target nucleic acids, and polynucleotides and compositions used for detecting target nucleic acids.
2. Description of the Related Art
Real-time polymerase chain reaction (RT-PCR) is a method used to monitor a process of amplification in real time whereby PCR products are amplified exponentially, and is one of the most commonly used methods in molecular biology. In particular, RT-PCR is used to monitor a process of producing PCR amplification products in real-time, to thereby quantify an amount of DNA in an original sample using an apparatus in which a thermal cycler and a fluorophotometer are integrated as one body.
The RT-PCR method may be divided into an absolute quantification analysis and a relative quantification analysis. The absolute quantification analysis is used to quantify the amount of DNA in a sample. After simultaneously performing qRT-PCR for a standard sample and an unknown sample, a standard curve may be generated from the standard sample. The amount of target DNA in the unknown sample can then be quantified from the standard curve. Generally, the standard curve (x-axis: Log10 (standard sample); y-axis: the number of cycle threshold) of the standard sample is ideal when a correlation index (R2) is about 1. Ideally, a slope derived from the standard curve is about −3.3 for a 1/10× diluted PCR product, and a slope is about −2.33 for a ⅕× diluted PCR product.
Currently, in most cases when a qRT-PCR is performed by a fluorescence method using probes such as Taqman™, a relatively short region of DNA is used as a target nucleic acid to maintain precise levels of nucleic acid quantification and detection. More time is needed for the elongation of longer target nucleic acids, however, and thus the detection and quantification of longer target nucleic acids of interest are less precise.
Accordingly, there is still a need for a method of detecting and quantifying long target nucleic acids with excellent precision and sensitivity, including instances when a conventional method such as RT-PCR are used.